A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G proteins) which, upon activation, relay signals to a variety of cellular effectors including at least four phospholipase C (PLC) variants and adenylyl cyclases.
G proteins mediate external signals by forming heterotrimers consisting of an alpha, beta and gamma subunit. Several isoforms of each subunit have been identified and therefore, through subunit heterogeneity, G proteins effectively integrate multiple signaling cascades. The alpha subunits of G proteins contain the GTP binding site and intrinsic catalytic GTPase activity. Based on sequence similarity and function, these subunits have been classified into four major groups; Gs, which stimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq, which activate PLC isoforms and G12/13, which mediate pathways associated with cell growth and differentiation (Hamm, J. Biol. Chem., 1998, 273, 669-672).
G-alpha-11 is a member of the Gq subfamily of G proteins whose primary function is to activate PLC-.beta. isoforms producing second messengers and affecting intracellular calcium stores. In the eye, for example, visual transduction involves PLC activity which results in calcium release and which is believed to be activated by light (Peng et al., Proc. Natl. Acad. Sci. U S A, 1997, 94, 1995-2000).
Only one PLC-.beta. isoform (PLC-.beta.4) is found in the retinal rod outer segment and studies using antibodies to Gq proteins demonstrated that G-alpha-11 is the only member of the Gq family found in this area of the retina. These results suggest that PLC-.beta.4 signaling is activated through G-alpha-11 (Peng et al., Proc. Natl. Acad. Sci. U S A, 1997, 94, 1995-2000).
By coupling to the ml muscarinic receptor and the .alpha.1-adrenoreceptors, G-alpha-11 affects intracellular calcium stores (Dippel et al., Proc. Natl. Acad. Sci. U S A, 1996, 93, 1391-1396; Macrez-Lepretre et al., J. Biol. Chem., 1997, 272, 5261-5268). In both studies, antisense oligonucleotides directed against G-alpha-11 were microinjected into the nucleus of cells in order to block G-alpha-11 expression. In rat basophilic leukemia cells overexpressing the ml muscarinic.receptor, antisense inhibition of G-alpha-11 resulted in the loss of the PLC-.beta.-induced calcium signal (Dippel et al., Proc. Natl. Acad. Sci. U S A, 1996, 93, 1391-1396). Likewise, antisense studies using the same G-alpha-11 oligonucleotide in rat portal vein myocytes demonstrated that G-alpha-11 was responsible for modulating intracellular store-dependent calcium entry (Macrez-Lepretre et al., J. Biol. Chem., 1997, 272, 5261-5268).
Single knockout mice have also been created that lack the Gq family members. All of the homozygous deficient mice were viable and fertile. However, mice lacking two Gq family members, both G-alpha-q and G-alpha-11, died during embryogenesis (Xu et al., J. Biol. Chem., 1998, 273, 27275-27279).
The G-alpha-11 protein, unlike other alpha subunits, contains a six amino acid insert which has been shown to be responsible for receptor coupling specificity (Kostenis et al., J. Biol. Chem., 1997, 272, 19107-19110), and mutations in this region produce constitutively active mutant proteins capable of coupling to multiple receptors (Kostenis et al., J. Biol. Chem., 1998, 273, 17886-17892). Using these mutant G-alpha-11 proteins, recent studies have shown that G-alpha-11 activates all three of the major MAPK pathways (Nagao et al., J. Biol. Chem., 1998, 273, 22892-22898; Yamauchi et al., J. Biol. Chem., 1997, 272, 27771-27777).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of G-alpha-11 and to date strategies aimed at inhibiting or investigating G-alpha-11 function have involved the use of antibodies, constitutively active forms of the protein, nuclear microinjection of antisense oligonucleotides and gene knock-outs in mice.
These strategies, however, are untested as therapeutic protocols. Consequently there remains a long felt need for additional agents capable of effectively inhibiting G-alpha-11 function and antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of G-alpha-11 expression.